By Victor

Protein kinases are enzymes which transfer phosphate groups from donors such as ATP to proteins in a process call “phosphorylation”. This process is usually reversible; phosphatases are the enzymes which “dephosphorylate” or remove phosphate groups from proteins. Each phosphate group carries two negative charges. The addition or removal of a phosphate group can change the three-dimensional shape of the protein. Such “conformational changes” alter the biological activity of the protein, changing the way it interacts with other proteins in a complex communication network within the cell. Many cellular receptors use phosphorylation as a means of signal transduction. For example, various receptors for growth factors, such as insulin-like growth factor (IGF) and epidermal growth factor (EGF), use phosphorylation to induce cellular growth and proliferation. Kinases and phosphatases can be thought of as “on” and “off” switches which are activated by such extracellular signaling molecules.

Cancer Treatment or Holy Grail?

In the 1980s it was observed that, unlike normal cells, many cancer cells proliferate in the absence of extracellular growth factors. Many tumors were found to overexpress EGF. Often phosphorylation signaling pathways were dysregulated, meaning that the “switch” was always stuck in the “on” position. (ref, ref) Kinase inhibition became a promising target to “turn off” these dysfunctional switches. Kinases are also involved in the replication of HIV and other pathogens, as well as virtually all physiological processes, including many pathways associated with aging (e.g. PI3-kinase, AKT, mTOR, MAPK, etc.) The prospect of being able to selectively modulate kinase activity seemed to hold the key for treating a wide-range of conditions. Thus, the quest began. It has been estimated that one-third of pharmaceutical company research efforts have been focused on kinase inhibition. Spurred on by the spectacular success of imatinib, the quest continues today.

Imatinib – the “Magic Bullet”.

In 2001, Imatinib (Gleevec) was featured on the cover of TIME magazine, as “the magic bullet” for treating cancer. In 2009, the developers received the prestigious Lasker Award (known as the “American Nobel Prize”) for “converting a fatal cancer into a manageable chronic condition”.(ref) Imatinib is a specific kinase inhibitor, which induces complete remission in the great majority of patients with chronic-phase CML, a common form of leukemia, with very few side effects. Imatinib, and its successors, have also been widely heralded as a model of rational drug design, using a targeted approach, and a new paradigm for the development of “next-generation” kinase inhibitors. (ref).

Taking a Closer Look.

It would appear that we are on the verge of a new age, not just in cancer treatment, but in pharmacological development. Imatinib was developed over a decade ago. The hype continues. But is it justified? The real question is whether the success of imatinib is repeatable with other conditions, or was it a special case. The answer is that the remarkable success of imatinib was a very exceptional case, unlikely to be repeated with very many other conditions.

Pathophysiology of CML:

“CML was the first malignancy to be linked to a clear genetic abnormality, the chromosomal translocation known as the Philadelphia chromosome. In this translocation, parts of two chromosomes (the 9th and 22nd by conventional karyotypic numbering) switch places. As a result, part of the BCR (“breakpoint cluster region”) gene from chromosome 22 is fused with the ABL gene on chromosome 9. This abnormal “fusion” gene generates a protein of p210 or sometimes p185 weight (p is a weight measure of cellular proteins in kDa). Because abl carries a domain that can add phosphate groups to tyrosine residues (a tyrosine kinase), the bcr-abl fusion gene product is also a tyrosine kinase.[1][6] The fused BCR-ABL protein interacts with the interleukin 3beta(c) receptor subunit. The BCR-ABL transcript is continuously active and does not require activation by other cellular messaging proteins. In turn, BCR-ABL activates a cascade of proteins which control the cell cycle, speeding up cell division. Moreover, the BCR-ABL protein inhibits DNA repair, causing genomic instability and making the cell more susceptible to developing further genetic abnormalities. The action of the BCR-ABL protein is the pathophysiologic cause of chronic myelogenous leukemia. With improved understanding of the nature of the BCR-ABL protein and its action as a tyrosine kinase, targeted therapies have been developed (the first of which was imatinib mesylate) which specifically inhibit the activity of the BCR-ABL protein. These tyrosine kinase inhibitors can induce complete remissions in CML, confirming the central importance of bcr-abl as the cause of CML.[6]”

CML is unique.

1. Very few conditions are the result of such a single genetic abnormality.
2. The BCR-ABL fusion protein represents the ideal molecular target.   Since it is an abnormal protein that does not ordinarily even exist in healthy humans, inhibiting its activity does not interfere with normal processes, or cause unwanted side effects.

Very few conditions are the result of a single well-known, abnormal, protein, which can be readily targeted without interfering with normal physiological processes. The success of imatinib, and similar compounds, does not represent a paradigm shift in drug development. On the contrary, the complexity of phosporylation networks will require robust systems biology approaches, not a single-target reductionist approach.

The Neglected Phosphatases

In the rush to investigate kinase inhibitors, the important role of the other “off” switch has been largely ignored. Just as dysregulated kinase activity is often associated with cancer, many phosphatases act as tumor suppressors (ref). In fact, mutations reducing phosphatase activity appear to play a much more important role in tumorigenesis than do mutations affecting kinase activity. One important phosphatase, PTEN, is the second most frequently mutated gene in human cancers, following p53. Given the fact that PTEN also regulates p53 levels (ref), some have even called PTEN “the new guardian of the genome”. (ref) For more information on p53, see: p53 and Longevity.

What has been the track record of target-based approaches?

Despite advances in our understanding of genomics, and the development of ever-more advanced methodological technology, the discovery of effective pharmaceuticals has declined.

How were new medicines discovered?

“Investment in drug research and development (R&D) has increased substantially in recent decades, but the annual number of truly innovative new medicines approved by the US Food and Drug Administration (FDA) has not increased accordingly, and attrition rates are very high¹. Indeed, in a recent analysis² it was noted that without a dramatic improvement in R&D productivity, the pharmaceutical industry cannot sustain sufficient innovation to replace the loss of revenues due to patent expirations for successful products. …Since the dawn of the genomics era in the 1990s, the main focus of drug discovery has been on drug targets, which are typically proteins that appear to have a key role in disease pathogenesis^{3, 4, 5}.Modification of target activity provides a rational basis for the discovery of new medicines; a target-centric approach provides a specific biological hypothesis to be tested and a starting point for the identification of molecules to do this with. Tremendous advances have been made in the development of new tools to identify targets and compounds that interact with these targets (for example, high-throughput target-based screening assays that are applicable to key protein families such as G protein-coupled receptors and kinases). Structure-based tools that can be used to aid lead identification and optimization for some targets have also been developed, including X-ray crystallography and computational modeling and screening (virtual screening).

However, despite the power of these tools to identify potential drug candidates, R&D productivity remains a crucial challenge for the pharmaceutical industry, which raises questions about the possible limitations of a target-centric approach to drug discovery…The increased reliance on hypothesis-driven target-based approaches in drug discovery has coincided with the sequencing of the human genome and an apparent belief by some that every target can provide the basis for a drug. As such, research across the pharmaceutical industry as well as academic institutions has increasingly focused on targets, arguably at the expense of the development of preclinical assays that translate more effectively into clinical effects in patients with a specific disease.”

Multiple Targets: Systems Pharmacology

Despite the disappointing track record of single-target approaches, I do not believe that target-based approaches should be abandoned. What is needed is a multiple-target approach, which can be combined with phenotypic assays. Whole systems approaches are needed to effectively model the interconnected complexity of human physiology. Such approaches will need to use methods discussed in Systems Biology and its tools. Protein phosphorylation networks comprise a complex system. Alterations can be compensated for in unexpected ways; and small indirect effects can have large unexpected consequences.

Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases

“Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery, and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis… Another finding of this study was the unexpectedly strong dominance of indirect effects (as opposed to direct molecular target effects), which were often without a resulting strong cellular phenotype. To some extent, this observation fits with a view of signaling networks having to be highly flexible and redundant to respond to an ever-changing environment while maintaining stable cellular states (44). This constrains the architecture of the system, as described by the “law of requisite variety” (45, 46), a fundamental law in systems control theory. It states that stable systems have to encode a number of control states that is higher than or equal to the number of states to be controlled. Considering that for each cell the space of “environmental states” is enormous, consequently, also the cellular “control variable space” must have an equal or greater size. The combinatorial possibilities of the phosphoproteome seem to ideally fulfill this demand (44).”

Conclusions

Clearly, kinase inhibition makes a very attractive pharmaceutical target. Many small-molecule inhibitors have been approved for cancer treatment. However, the current pharmaceutical company obsession with single-target, kinase-inhibiting drugs highlights the problem of too much research being driven by the quick profit-incentive, at the expensive of truly understanding the dynamics of the underlying physiological processes. Since kinases have such a wide-range of effects in multiple tissues types, simply inhibiting them is likely to have many detrimental off-target effects. We, really, first need to better understand what is happening at the molecular and organismal level in these signaling pathways in order to increase therapeutic efficacy and specificity. Gone are the days in which a single researcher, or small group, could enter a laboratory and develop breakthrough results. Future advances will require greater interdisciplinary collaboration, relying on teams with experts in many different fields, such as physics, genetics, enzymology, mathematics, proteomics, etc. Such multidisciplinary, whole systems approaches will be challenging, but necessary to make effective use of currently available technology, and to meaningfully interpret the resulting datasets, in order to take our understanding of complex biological processes, and the development of therapeutics to the next level.

See also:

Targeting the cancer kinome through polypharmacology

Systems approaches to polypharmacology and drug discovery

Network analyses in systems pharmacology

Role of systems pharmacology in understanding drug adverse events

 

By Vince Giuliano

This blog entry is about somatic stem cells, the natural kind that reside in adult bodies, the factors that affect their health and longevity, the changes they undergo in the process of aging, and the roles they possibly play in overall human aging.  Further, it outlines how epigenetic interventions in such stem cells could possibly contribute to longer human lifespans. In the interest of presenting a comprehensive overview, I review previously-reported findings as well as several newer ones.

Background

Somatic stem cells, also known as adult stem cells are multipotent cells, that is, a type of somatic stem cell that can differentiate into cells belonging to several different related cell lineages but not into all ultimate body cell types.  They generally live in stem cell niches, protective microenvironments in the body unique to the kind of somatic stem cell involved.  Important types of somatic stem cells include:
·        Hematopoietic stem cells (HSCs) which are “multipotent stem cells that give rise to all the blood cell types from the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells)(ref).
·        Mesenchymal stem cells, or MSCs, which are “multipotent stem cells that can differentiate into a variety of cell types,^[1] including: osteoblasts (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells)(ref).”
·        Endothelial stem cells.  “Endothelial Stem Cells are one of the three types of Multipotent stem cells found in the bone marrow. They are a rare and controversial group with the ability to differentiate into endothelial cells, the cells which line blood vessels(ref).

Also there are Mammary stem cells, Neural stem cells, Olfactory adult stem cells, Neural crest stem cells and Testicular cells.

Adult stem cells belong to a major category of cells in what I have called the stem cell supply chain.  In my treatise I related: “In a simplified model, think of the 210 kinds of cells found in the human body as falling in five categories:

A. Pluripotent cells, ones which are and capable of differentiating into any other cells. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are in this category,

B. Relatively undifferentiated multipotent somatic stem cells, such as may exist in bone marrow or vascular walls (e.g. hematopoietic stem cells, mesenchymal stem cells and pericytes). These multipotent adult stem cells are each capable of differentiating into a variety of kinds of somatic cells.

C. More differentiated stem and progenitor cells (e.g. endothelial progenitor cells, myoblasts or satellite cells in muscle tissue). These are cells capable of differentiating only into more-specific somatic cell types.

D. Normal body somatic cells (e.g. cardiomyocytes, red blood cells, leukocytes, keratinocytes, melanocytes, and Langerhans cells).

E. Senescent cells, ones which no longer can divide.

The list is in order of increasing cell-type specificity and decreasing potency to differentiate into other cell types. Starting at conception and throughout life, all cells on this list except the senescent ones will selectively reproduce and possibly differentiate into cells of types further down in the list.”

Adult stem cells of a given type under conditions of youth and health typically differentiate to produce a defined mix of daughter cell types.  For example, hematopoietic stem cells (HSCs) “give rise to all the blood cell types from the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells(ref).”  As discussed below, aged or damaged adult stem cells may give rise to a skewed mix of daughter cells.

There are a few key topics that I do not treat here though to some extent they have been discussed in past blog entries, including disease therapies based on use of adult stem cells and practical dietary and lifestyle interventions that can contribute to adult stem cell health.  Without doubt, these will be discussed further in future blog entries.

I organize this post by major observations, attempting to be fairly comprehensive in coverage.  Some of the observations are quite basic and I have written about them before in my treatise and in multiple blog entries.  I will cite relevant past writings as they apply to such observations.  Other key observations are based on research published only in the last six months.

Overview

The 2011 publication Manifestations and mechanisms of stem cell aging reports “Adult stem cells exist in most mammalian organs and tissues and are indispensable for normal tissue homeostasis and repair. In most tissues, there is an age-related decline in stem cell functionality but not a depletion of stem cells. Such functional changes reflect deleterious effects of age on the genome, epigenome, some of which arise cell autonomously and others of which are imposed by an age-related change in the local milieu or systemic environment. Notably, some of the changes, particularly epigenomic and proteomic, are potentially reversible, and both environmental and genetic interventions can result in the rejuvenation of aged stem cells. Such findings have profound implications for the stem cell–based therapy of age-related diseases.”

Health and longevity of somatic stem cells is critical for human organismal health and longevity

This point has been known for some time and is discussed in the Section on the 14^th theory of aging in my treatise ANTI-AGING FIREWALLS – THE SCIENCE AND TECHNOLOGY OF LONGEVITY. “Health for older people requires continuing operation of the stem cell supply chain at some levels throughout life. If an injury is sustained, mesenchymal stem cells must make new tissue cells. If there is loss of blood, hematopoietic stem cells must make new blood cells. And cells that die of attrition trauma or apoptosis must be replaced by new ones. “Hematopoietic stem cells (HSCs) are responsible for blood cell production throughout the lifetime of an individual(ref),” and the same is true for other types of stem cells. A new concept is emerging: that age-related changes in the stem cells in many body organs may be responsible for deterioration and decline in functionality of those organs. As a simple example, new research suggests that gray or white hair is due to age-related depletion of melanocytes which is a direct result of depletion of melanocyte stem-cells(MSCs) which in turn is the result of DNA damage. It has been known for some time that ” – hair graying is caused by defective self-maintenance of MSCs(ref).” These stem cells, living in hair follicles, can normally both reproduce making new stem cells and differentiate into mature color-producing melanocytes. The new research based on experimentation with mice suggests that DNA damage to MSCs causes them to stop reproducing and instead terminally differentiate into melanocytes. As the melanocytes in hair follicles die off, there are no new melanocytes to replace them because there are no more MSCs to make them.”

“As multicellular organisms age, there is a gradual loss of tissue homeostasis and organ function. Throughout life, populations of adult stem cells maintain many tissues, such as the blood, skin and intestinal epithelium. Therefore, it is likely that the decrease in tissue homeostasis can be attributed to an age-related decline in the ability of stem cells to replace damaged cells. Although cell autonomous changes occur as the organism ages that result in the inability of stem cells to proliferate or self-renew, or of daughter cells to differentiate along a specific lineage, local and systemic changes can also affect the ability of stem and progenitor cells to function properly (Energy metabolism in adult neural stem cell fate 2011).”

The 2011 review article Manifestations and mechanisms of stem cell aging relates “Aging is accompanied by a decline in the homeostatic and regenerative capacity of all tissues and organs (Kirkwood, 2005; Rando, 2006). With age, wound healing is slower in the skin, hair turns gray or is lost, skeletal muscle mass and strength decrease, the ratio of cellular constituents in the blood is skewed, and there is a decline in neurogenesis (Sharpless and DePinho, 2007). As the homeostatic and regenerative activities of these tissues are attributable to the resident stem cells, these age-related changes are reflections of declines in stem cell function (Bell and Van Zant, 2004; Dorshkind et al., 2009; Jones and Rando, 2011). Clearly, in terms of organismal aging, the focus on stem cells is most relevant for those tissues in which normal cellular turnover is very high, such as epithelia of the skin and gut, as opposed to tissues, such as the cerebral cortex and the heart, in which cellular turnover in adults is exceedingly low (Rando, 2006). There is also an increasing interest in the therapeutic potential of stem cells to treat age-related degenerative diseases or conditions, further highlighting the importance of understanding the relationship between stem cell function and the properties of aged tissues. Within this context, it is essential to understand how the local environment influences stem cells, how aging affects stem cell number and function, and the extent to which aspects of stem cell aging may be reversible.”

Stem cells reside in niches and there is a close interplay between stem cells and their niches in determining stem cell health, their differentiation capabilities and their fates.

Again from my treatise “Adult stem cells live in niches – stem cell microenvironments and the health of the stem cells and their ability to reproduce or differentiate both depend upon and condition the states of their niches. The behavior of stem cells can be expected to be very different within and without their niches.  “Interaction of HSCs with their particular microenvironments, known as stem cell niches, is critical for maintaining stem cell properties, including self-renewal capability and ability for differentiation into single and multiple lineages. In the niche, the niche cells produce signaling molecules, extracellular matrix, and cell adhesion molecules and regulate stem cell fates(ref).” “Various niche factors act on embryonic stem cells to alter gene expression, and induce their proliferation or differentiation for the development of the fetus. Within the human body, stem cell niches maintain adult stem cells in a quiescent state, but after tissue injury, the surrounding microenvironment actively signals to stem cells to either promote self renewal or differentiation to form new tissues(ref).” For example, “Haematopoietic stem-progenitor cells (HSPCs) reside in the bone marrow niche, where interactions with osteoblasts provide essential cues for their proliferation and survival(ref).” Among the other places where niches of adult stem cells can be found are hair follicles (see the blog entry Hair stem cells) and in dental pulp (see the blog entry Dental pulp stem cells).”

Adult stem cells are subject to aging and stock depletion like other dividing cells types

In general, adult stem cells either divide like normal body cells do (mitosis) or differentiate in which case a stem cell produces another like stem cell and a progenitor cell which further differentiates and divides to make normal somatic body cells. “Differentiation dramatically changes a cell’s size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are largely due to highly-controlled modifications in gene expression. With a few exceptions, cellular differentiation almost never involves a change in the DNA sequence itself(ref).”  In other words, differentiation involves a significant shift in epigenetic state of a cell to a more specific less-potent status.

“Adult stem cells are exposed to many of the same factors that lead to age-related changes in their replicative or postmitotic progeny, but stem cells must resist those changes as a self-renewing population to assure proper function and normal tissue homeostasis across the lifespan (Rando, 2006; Sharpless and DePinho, 2007; Jones and Rando, 2011). As a replicative population that may have prolonged periods of quiescence (Fig. 1), stem cells must possess defense and repair mechanisms that are relevant to both highly proliferative cells and to long-lived postmitotic cells (Rando, 2006) — During prolonged periods of quiescence and by the process of self-renewal to establish a cellular continuum, stem cells experience chronological aging caused by the accumulation of damaged or aberrant intracellular molecules. During the process of asymmetric cell division and self-renewal, stem cells also experience replicative aging, which is particularly important in tissues with high turnover rates.  — In long-lived animals, adult stem cells, particularly those in continuously renewing tissues, undergo many rounds of cell division to maintain normal tissue homeostasis (Fuchs et al., 2001; van der Flier and Clevers, 2009). During each round of DNA replication, processes that underlie replicative aging, including telomere shortening, chromosome rearrangements, and single base mutations (Ben-Porath and Weinberg, 2005), can occur and ultimately lead to cellular senescence (Hayflick, 1965; Campisi and d’Adda di Fagagna, 2007). Experimental manipulations, such as serial transplantation, clearly reveal that adult stem cells have a finite replicative lifespan that can be exhausted (Siminovitch et al., 1964; Waterstrat and Van Zant, 2009). However, as serial transplantation experiments subject stem cells to excessive rounds of cell division, it remains to be determined whether replicative aging alone is sufficient to contribute to the decline of stem cell function in long-lived mammals during normal aging(ref).”

Continuing(ref): “Adult stem cells are also susceptible to the kinds of age-related changes, namely chronological aging, that occur in nondividing cells, such as neurons and cardiomyocytes (Busuttil et al., 2007). These changes include the accumulation of damaged macromolecules, such as proteins, lipids, and nucleic acids, some of which may, in fact, aggregate and form stable, long-lived complexes that are toxic to the cell (Rajawat et al., 2009; Koga et al., 2011). Adult stem cells exhibit prolonged periods of quiescence in most mammalian tissues (Li and Clevers, 2010). Damaged macromolecules can accumulate in stem cells during this time, just as in long-lived postmitotic cells. Specific macromolecules or macromolecular aggregates may even be selectively retained in stem cells as they undergo the process of self-renewal by asymmetric cell division (Conboy et al., 2007; Knoblich, 2008). In this sense, the self-renewing progeny represent a kind of cellular continuum and only add to the risk that adult stem cells may suffer from the effects of chronological aging(ref).”

In mammals, the pools of available adult stem cells do not normally run out with aging, but aged adult stem cells may become resistant to differentiation and may not differentiate with the correct mix of end cell types

“Aging in stem cells causes changes in the fate or functionality of stem cell progeny. In some cases, such as neural stem cells (NSCs) and melanocyte stem cells (Maslov et al., 2004; Inomata et al., 2009), these changes may lead to a depletion of the stem cell pool (Fig. 2; Kuhn et al., 1996; Maslov et al., 2004). However, in most stem cell compartments, the number of stem cells does not decline significantly with age (Booth and Potten, 2000; Brack and Rando, 2007; Giangreco et al., 2008); rather, these stem cells experience a change in cell fate with age. — In young animals, stem cells divide asymmetrically to self-renew and give rise to lineage-specific differentiated progeny during tissue homeostasis or regeneration. With age, some stem cells lose their lineage specificity and give rise to nonfunctional progeny, resulting in loss of tissue integrity and decline of physiological function, even though the number of stem cells remains unaffected. Some stem cells lose the capacity for self-renewal, resulting in symmetric cell divisions giving rise to two differentiated daughters and a gradual depletion of the stem cell pool. The senescence of stem cells can also contribute to a loss of functional stem cells. The increase in malignancies with age, particularly in epithelia with high turnover rates, has been proposed to arise from within the stem cell compartment or from early progenitors(ref).”

Continuing: “Within the hematopoietic system, the ratios of differentiated progeny change with age. Hematopoietic stem cells (HSCs) from both old humans and old mice show an increased propensity to differentiate along the myeloid rather than the lymphoid lineage (Sudo et al., 2000; Rossi et al., 2005). Such lineage bias is not caused by a change in the differentiation potential of individual HSCs but rather by a preferential selection of distinct subsets of HSCs over time (Cho et al., 2008; Beerman et al., 2010; Challen et al., 2010). The differential responsiveness of these two HSC populations to TGF-β may further enhance the skewed ratio between myeloid versus lymphoid progeny in old individuals (Challen et al., 2010). Although the progeny of the aged HSCs do not include any cells that are not otherwise part of the normal repertoire of cells produced by HSCs, this lineage skewing results in a decreased number of memory B cells and naive T cells (Linton and Dorshkind, 2004; Min et al., 2004) and adversely affects immunological responses (Rink et al., 1998; Grubeck-Loebenstein et al., 2009.”

Adult stem and progenitor cells are subject to replicative senescence and express very different genes when young and old, but not due to telomere erosion

This point was made in the October 2010 blog entry Telomere lengths, Part 3: Selected current research on telomere-related signaling. “The 2009 publication Aging and Replicative Senescence Have Related Effects on Human Stem and Progenitor Cells is important in that a) it established that at least some stem cells are subject to replicative senescence, b) gene expression patterns of young and old stem cells vary drastically with age, and that c) telomere erosion does not appear to be responsible for the differences in gene expression of old and younger stem cells. The research looked at the gene-expression effects of replicative senescence on mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC) and compared these to the gene-expression effects found in in-vivo aging. It found the effects to be similar, suggesting that stem and progenitor cells are subject to replicative senescence, just as other types of body cells are. Further, at least in HPCs, telomere erosion does not appear to be well-correlated with aging.”

“The same publication reports “The regenerative potential diminishes with age and this has been ascribed to functional impairments of adult stem cells. Cells in culture undergo senescence after a certain number of cell divisions whereby the cells enlarge and finally stop proliferation. This observation of replicative senescence has been extrapolated to somatic stem cells in vivo and might reflect the aging process of the whole organism. In this study we have analyzed the effect of aging on gene expression profiles of human mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC). MSC were isolated from bone marrow of donors between 21 and 92 years old. 67 genes were age-induced and 60 were age-repressed. HPC were isolated from cord blood or from mobilized peripheral blood of donors between 27 and 73 years and 432 genes were age-induced and 495 were age-repressed. The overlap of age-associated differential gene expression in HPC and MSC was moderate. However, it was striking that several age-related gene expression changes in both MSC and HPC were also differentially expressed upon replicative senescence of MSC in vitro. Especially genes involved in genomic integrity and regulation of transcription were age-repressed. Although telomerase activity and telomere length varied in HPC particularly from older donors, an age-dependent decline was not significant arguing against telomere exhaustion as being causal for the aging phenotype. These studies have demonstrated that aging causes gene expression changes in human MSC and HPC that vary between the two different cell types. Changes upon aging of MSC and HPC are related to those of replicative senescence of MSC in vitro and this indicates that our stem and progenitor cells undergo a similar process also in vivo.”

Age-related behavior of adult stem cells is influenced by epigenetic factors that impact on key signaling pathways involved in cell division and differentiation

“The ability of stem cells to produce an appropriate repertoire of tissue-specific progeny is crucial for functional tissue homeostasis and regeneration. The extent to which adult stem cells and their progeny are committed to a particular lineage is determined largely by the epigenome, influencing which genes will be expressed and which will be repressed and ultimately shaping the phenotypic characteristics of the cells (Bernstein et al., 2006; Mikkelsen et al., 2007; Hemberger et al., 2009). The execution of the epigenomic program that influences the fate of stem cell progeny is modulated by environmental factors and mediated by signaling pathways that have important roles in organogenesis during development, including the Wnt, Notch, and Hedgehog pathways (Berger, 2007; Brack et al., 2008; Rittié et al., 2009; van der Flier and Clevers, 2009). With age, untimely activation of these pathways as a result of signals from the “old environment” may lead to aberrant lineage specification of stem cell progeny as has been demonstrated in tissues, such as skeletal muscle, tendon, and the hematopoietic system (Sudo et al., 2000; Taylor-Jones et al., 2002; Brack et al., 2007; Zhou et al., 2010). Accumulation of these abnormal progeny contributes to the gradual deterioration of tissue structure and function associated with aging(ref).”

Notch and MAPK are key pathways involved in cell proliferation and differentiation

I first touched on these signaling pathways in the 2009 blog post Niche, Notch and Nudge.  “The grist of this post deals with both new research and a couple of complicated cell signal-transduction pathways that have been extensively studied for over 15 years now, known as Notch and MAPK. — Notch is an ancient signaling pathway that has been inherited from primitive multi-cellular organisms and has to do with signaling between cells, such as when stem cells decide to differentiate. “Because Notch often acts in concert with other signaling pathways, it is able to regulate a diverse set of biological processes in a cell-context dependent manner(ref). “ Notch protein receptors (there are 4 different ones) sit on the surfaces of cells and communicate between adjacent cells via Notch ligands. Ligand binding to a receptor alters the chemical conformation, that is the three dimensional shape of the receptor protein(ref).” Intracellular proteins transmit Notch signals into the cell’s nucleus where they can activate genes, including ones that initiate differentiation in stem cells. Notch signaling can play an important role in determining the morphology of organs. For example see this publication. Also Notch plays several important roles in stem and progenitor cell differentiation, particularly ones that maintain balance during development. “Notch signaling is a powerful means of turning adult CNS precursor cells into astrocytes(ref).” “In the developing nervous system, the balance between proliferation and differentiation is critical to generate the appropriate numbers and types of neurons and glia. Notch signaling maintains the progenitor pool throughout this process(ref).”

MAPK/ERK is another very complicated signal transduction pathway way that couples intracellular responses to the binding of growth factors to cell surface receptors. MAPK signaling is important for cell growth and differentiation, inflammation and apoptosis. A diagram showing all the ways MAPK signaling can work would fill a large wall. For example this diagram shows four different MAPK cascades. Clicking on the individual bubbles in the diagram reveal more-detailed diagrams, showing cascades such as for growth, differentiation and inflammation.

Both Notch and MAPK signaling are deeply involved in embryogenesis and stem cell differentiation. It is no surprise that there is crosstalk between the Notch and MAPK pathways. For example, this report states: “Here we show that Notch signaling activation in C2C12 cells suppresses the activity of p38 MAPK to inhibit myogenesis. Our results show that Notch specifically induces expression of MKP-1, a member of the dual-specificity MAPK phosphatase, which directly inactivates p38 to negatively regulate C2C12 myogenesis.”

The same 2009 blog entry cites a number of other publications relating Notch and MAPK signaling to stem cell differentiation.

The protein JDP2 is involved with epigenetic modifications to histones relevant to age-related changes in stem cell differentiation and cell senescence

This subject is covered in the February 2011 blog post JDP2 – linking epigenetic modifications, stem cell differentiation, cell senescence, cell stress response, and aging.  “JDP2 is involved with epigenetic modifications to histones relevant to age-related changes in stem cell differentiation and cell senescence. Like the previously-discussed Smurf2 gene, JDP2 is involved in the regulation of the differentiation and proliferation of cells. Its presence or absence affects whether cells differentiate or become senescent. The new research has implications related to organismal aging and for the Programmed Epigenomic Changes theory of aging. I review some of the new publications here and relate new findings to matters I have discussed previously.”

Molecular control inhibiting differentiation, particularly the presence of P21 and adequate DNA methylation, is essential to prevent exhaustion of adult stem cell pools

This is an older finding pointed out in a May 2010 blog entry Something new about P21.  “Expression of P21 is a barrier to stem cell differentiation. The 2000 publication Hematopoietic Stem Cell Quiescence Maintained by p21^cip1/waf1 states “Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death.” In the absence of P21, hematopoietic stem cells would not remain quiescent in their niches but would instead prematurely differentiate when stress occurs exhausting the pools of those cells and interrupting the normal functioning of the stem cell supply chain leading to premature death. The 2009 paper Accelerating stem cell proliferation by down-regulation of cell cycle regulator p21 offers a consistent message. “Inhibition of the cell cycle regulator p21 results in significant acceleration of mesenchymal stem cell proliferation without promoting spontaneous cellular differentiation.”  That blog entry also describes how P21 control of differentiation is a key factor in the process of limb regeneration.

Adequate DNA methylation is also implicated in maintenance of pools of undifferentiated adult stem cells.  From the blog entry DNA Methyltransferases, stem cell proliferation and differentiation: “Adult stem cells, including neural progenitor cells and hematopoietic stem cells depend on DNA methylation for their survival in undifferentiated state. This methylation in turn depends critically on the actions of DNA methyltransferases. In plain language, the methyltransferases keep lineages of adult stem cells continuing in their niches throughout life instead of having all the adult cells differentiating early in life leaving no reserves of such cells.

Neurogenesis is a much-studied model of adult stem cell differentiation

Brain cell renewal depends on neurogenesis due to differentiation of neural adult stem cells mainly in the hippocampus and cell migration. The process goes on throughout life.  Neurogenesis is an important special case of adult stem cell differentiation.  What is known about neurogenesis some extent applies also to differentiation of other adult stem cell types.  See the blog entry Age-related memory and brain functioning – focus on the hippocampus.

Neural stem cell self-renewal and proliferation can be affected by epigenetic interventions in histone H2AX

The 2009 publication Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells reports “Adult neural stem cell proliferation is dynamic and has the potential for massive self-renewal yet undergoes limited cell division in vivo. Here, we report an epigenetic mechanism regulating proliferation and self-renewal. The recruitment of the PI3K-related kinase signaling pathway and histone H2AX phosphorylation following GABAA receptor activation limits subventricular zone proliferation. As a result, NSC self-renewal and niche size is dynamic and can be directly modulated in both directions pharmacologically or by genetically targeting H2AX activation. Surprisingly, changes in proliferation have long-lasting consequences on stem cell numbers, niche size, and neuronal output. These results establish a mechanism that continuously limits proliferation and demonstrates its impact on adult neurogenesis. Such homeostatic suppression of NSC proliferation may contribute to the limited self-repair capacity of the damaged brain.”

Multiple kinds of changes may be involved in older adult stem cells affecting their differentiation capabilities

Quoting selectively from the 2011 publication Manifestations and mechanisms of stem cell aging “Among the cell-intrinsic changes that may mediate age-related changes in stem cell function are alterations at the level at the genome, the epigenome, and the proteome.

• Genome-level changes may include including single- and double-strand DNA breaks, chromosomal translocations, telomere shortening, and single base mutations (Akbari and Krokan, 2008; Wang et al., 2009). DNA repair systems have evolved to maintain genomic integrity, and it has been proposed that the intrinsic DNA repair activity and fidelity in different species may influence the rate of aging (Hart and Setlow, 1974). Mutations in proteins involved in DNA repair, such as the WRN (Werner Syndrome ATP-Dependent) helicase and the ATM (Ataxia Telangiectasia Mutated) kinase, have been associated with segmental progeroid syndromes in humans and mice that have features of accelerated aging in multiple tissues and organs (Savitsky et al., 1995; Gray et al., 1997; Kudlow et al., 2007), providing evidence for the crucial role of DNA repair machinery for normal tissue homeostasis.
• Unlike acquired DNA mutations, epigenomic changes, including DNA methylation and posttranslational modifications of histones, are dynamically maintained by a balance among chromatin-remodeling complexes and are, thus, reversible (Goldberg et al., 2007). Given the influence of cell extrinsic factors on the epigenome and the reversibility of chromatin modifications, epigenomic changes may underlie the stochastic aspects of aging (Herndon et al., 2002; Fraga et al., 2005; Kirkwood, 2005) and certain environmental influences that delay or even apparently reverse aging, such as the lifespan-extending effect of dietary restriction and the rejuvenation of aged stem cells by exposure to a young environment (Conboy et al., 2005; Dorshkind et al., 2009; Fontana et al., 2010). In yeast, lifespan extension by dietary restriction appears to require Sir2 (Lin et al., 2000), a histone deacetylase that has been shown to extend the lifespan in several model organisms (Longo and Kennedy, 2006). In Caenorhabditis elegans, members of the H3K4 methyltransferase complex affect lifespan in a germline-dependent manner (Greer et al., 2010).
• Maintenance of the intracellular proteome requires timely removal of improperly folded or damaged proteins that can otherwise impede normal cellular function (Koga et al., 2011). Autophagosomes, chaperones, lysosomes, and the ubiquitin–proteasome system are all important cellular processes and machineries that maintain protein homeostasis (Rajawat et al., 2009; Koga et al., 2011). Together, they sense and remove misfolded or aberrant proteins in cells and ensure a functional proteome. With age, the protein homeostatic machinery becomes less efficient and less effective (Rodriguez et al., 2010; Koga et al., 2011), and these functional declines would only accentuate the negative effect of proteomic changes during aging. — Age-related increases in the levels of damaged proteins have been well documented in long-lived postmitotic cells, such as neurons, cardiomyocytes, and skeletal myofibers, and in some cases, these damaged proteins form aggregates or inclusion bodies that can cause proteotoxicity to cells (Rodriguez et al., 2010.”

As pointed out in my treatise “Buildup of levels of Ink4a/P16 associated with aging slows down the rate of differentiation of adult stem cells. “Recent evidence shows that loss of Bmi-1, a polycomb transcriptional repressor of theInk4a-Arf locus, results in progressive loss of HSCs in adult mice with subsequent failure of hematopoiesis.” – “ These results show that either both p16Ink4a and p19Arf can inhibit HSC self-renewal in a serial transplant setting, or that only p16Ink4a is necessary(ref).“

Also I mention that in Victor’s blog entry P53 and Longevity, there is some  discussion of the role of P53 with respect to stem cells.  “There is an intimate relationship among p53, stem cell development, and epigenetic regulation of these processes, and it began to evolve in the fishes.” (ref).

Among the key factors affecting adult stem cells and their ability to differentiate are aging-related changes in the niches. As pointed out in the blog entry What every vampire already knows, “age-related loss of capability to reproduce and differentiate has to do with what is going on in the niches in which stem cells live. “Our results reveal that aged differentiated niches dominantly inhibit the expression of Oct4 in hESCs and Myf-5 in activated satellite cells, and reduce proliferation and myogenic differentiation of both embryonic and tissue-specific adult stem cells (ASCs). Therefore, despite their general neoorganogenesis potential, the ability of hESCs, and the more differentiated myogenic ASCs to contribute to tissue repair in the old will be greatly restricted due to the conserved inhibitory influence of aged differentiated niches(ref).”

Energy metabolism is critical in determining stem cell health and fate

The point is made for neural stem cells in the 2011 publication Energy metabolism in adult neural stem cell fate.  “The adult mammalian brain contains a population of neural stem cells that can give rise to neurons, astrocytes, and oligodendrocytes and are thought to be involved in certain forms of memory, behavior, and brain injury repair. Neural stem cell properties, such as self-renewal and multipotency, are modulated by both cell-intrinsic and cell-extrinsic factors. Emerging evidence suggests that energy metabolism is an important regulator of neural stem cell function. Molecules and signaling pathways that sense and influence energy metabolism, including insulin/insulin-like growth factor I (IGF-1)-FoxO and insulin/IGF-1-mTOR signaling, AMP-activated protein kinase (AMPK), SIRT1, and hypoxia-inducible factors, are now implicated in neural stem cell biology. Furthermore, these signaling modules are likely to cooperate with other pathways involved in stem cell maintenance and differentiation. This review summarizes the current understanding of how cellular and systemic energy metabolism regulate neural stem cell fate. The known consequences of dietary restriction, exercise, aging, and pathologies with deregulated energy metabolism for neural stem cells and their differentiated progeny will also be discussed. A better understanding of how neural stem cells are influenced by changes in energy availability will help unravel the complex nature of neural stem cell biology in both the normal and diseased state.”  Note that each of the pathways mentioned here are known to be involved in organismal aging.

The reduced functional and differentiation capabilities of older adult stem can to some extent  be rejuvenated by epigenetic interventions

The 2011 publication Epigenetic regulation of aging stem cells relates “The function of adult tissue-specific stem cells declines with age, which may contribute to the physiological decline in tissue homeostasis and the increased risk of neoplasm during aging. Old stem cells can be ‘rejuvenated’ by environmental stimuli in some cases, raising the possibility that a subset of age-dependent stem cell changes is regulated by reversible mechanisms. Epigenetic regulators are good candidates for such mechanisms, as they provide a versatile checkpoint to mediate plastic changes in gene expression and have recently been found to control organismal longevity. Here, we review the importance of chromatin regulation in adult stem cell compartments. We particularly focus on the roles of chromatin-modifying complexes and transcription factors that directly impact chromatin in aging stem cells. Understanding the regulation of chromatin states in adult stem cells is likely to have important implications for identifying avenues to maintain the homeostatic balance between sustained function and neoplastic transformation of aging stem cells.”

The April 2010 blog entry DNA Methyltransferases, stem cell proliferation and differentiation reviews research of DNA methyltransferases and their key regulatory roles on the epigenetics of adult stem cells. “DNA methylation, particularly when applied to CG-rich promoter sequences, has been shown to silence gene expression in a heritable manner. DNA methylation is therefore a form of cellular memory. Because DNA methylation is not encoded in the DNA sequence itself, it is called an epigenetic modification (ref).”, I remind my readers that the 13^th theory of aging covered in my treatise, Programmed Epigenomic Changes, envisages aging as a systematically articulated set of epigenomic changes including changes in DNA methylation in cells accumulated with aging. See my blog entry Homicide by DNA methylation.” – That blog entry reviews research relating to how DNA methyltransferases 1. initiate and maintain methyl marks, 2. are involved in self-renewal of embryonic stem (ES) cells, and 3. act in somatic (adult) stem cells including: hematopoietic, epithelial, neural and muscle cells. It also relates to the molecular factors that keep stem cells from differentiating and the role of methyltransferases once those cells start differentiating.

Addressing adult stem cell aging may be a fruitful approach to addressing human aging

The 2011 publication Emerging models and paradigms for stem cell ageing reports “The interesting overlap between the biology of ageing and the biology of stem cells has been reviewed extensively^{3, 5, 6, 7, 8}. To the extent that stem cell ageing is itself an important factor in organismal ageing, it may be possible to develop therapeutic approaches to age-related diseases based on interventions to delay, prevent or even reverse stem cell ageing. Therefore, understanding the basic properties of stem cells as they age, and the mechanisms that promote or prevent stem cell ageing, have significant implications for regenerative medicine and the goal of extending ‘healthspan’.”

Dental pulp niches appear to be important both for dental health and possibly also for health and longevity.

The 2011 publication Dental pulp stem cells, niches, and notch signaling in tooth injury reports “Stem cells guarantee tissue repair and regeneration throughout life. The decision between cell self-renewal and differentiation is influenced by a specialized microenvironment called the ‘stem cell niche’. In the tooth, stem cell niches are formed at specific anatomic locations of the dental pulp. The microenvironment of these niches regulates how dental pulp stem cell populations participate in tissue maintenance, repair, and regeneration. Signaling molecules such as Notch proteins are important regulators of stem cell function, with various capacities to induce proliferation or differentiation. Dental injuries often lead to odontoblast apoptosis, which triggers activation of dental pulp stem cells followed by their proliferation, migration, and differentiation into odontoblast-like cells, which elaborate a reparative dentin. Better knowledge of the regulation of dental pulp stem cells within their niches in pathological conditions will aid in the development of novel treatments for dental tissue repair and regeneration.”  See also the blog entry Dental Pulp Stem Cells – the big needle vs the tooth fairy.

Targeting adult cancer stem cells is a form of therapy being actively researched.

This is a subject I have discussed before in the blog entries, and is not something I will get into further in this blog entry.  Recent relevant publications include:

Cancer stem cells and malignant gliomas. From pathophysiology to targeted molecular therapy (2011)

Hematopoietic stem cell niche is a potential therapeutic target for bone metastatic tumors (2011)

Additional relevant publications include:

Distinct Roles of Bcl-2 and Bcl-Xl in the Apoptosis of Human Bone Marrow Mesenchymal Stem Cells during Differentiation (2011)

MicroRNA – a contributor to age-associated neural stem cell dysfunction? (2011)

The microRNA cluster miR-106b~25 regulates adult neural stem/progenitor cell proliferation and neuronal differentiation (2011)

The p53 tumor suppressor protein regulates hematopoietic stem cell fate (2011)

MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxP1 levels in developing spinal cords (2011)

p73alpha regulates the sensitivity of bone marrow mesenchymal stem cells to DNA damage agents (2010)

FoxO3 regulates neural stem cell homeostasis (2009)

A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination (2009)

Genotoxic stress abrogates renewal of melanocyte stem cells by triggering their differentiation (2009)

 

 

 

 

By Vince Giuliano

The last seven months have seen a great deal of research related to and induced pluripotent stem cells (iPSCs) and cell reprogramming. This blog post is devoted to some important perspectives that have emerged resulting from that research.  A subsequent blog entry will be devoted to the topic of longevity of stem cells and the roles stem cells play in human longevity.

Background

The February 2011 publication Genomic instability in iPS: time for a break (Blasco et al) reports “Since their discovery, manuscripts characterizing properties of induced Pluripotent Stem (iPS) have flooded the literature. Among others, the analysis of the transcriptome and epigenome of iPS is now a recurrent theme that is helping to understand the molecular mechanisms behind reprogramming. Recent works have revealed that transcriptional and epigenetic reprogramming is often incomplete, which has raised some concerns on the nature of iPS. Inevitably, now the genome itself of iPS has been scrutinized; and the reports come with an unexpected twist: the presence of mutations in the genome of iPS. —.”

Continuing: “The term iPS was officially born, and has arguably become one of the fastest moving fields in biomedical research. However, a careful look at the original protocol raised the concern that one of the four factors included in the reprogramming cocktail was a well-known oncogene (Myc). In addition, reprogramming can also be stimulated by the presence of other oncogenes such as SV40 large T antigen (Mali et al, 2008) or by the loss of tumour suppressors like p53 or Arf (Menendez et al, 2010). To further fuel the concerns, developmental problems and tumours were reported in mice derived from iPS (Okita et al, 2007; Zhao et al, 2010). As a consequence, much of the recent works on iPS have been dedicated to the development of safer protocols such as defining an even more minimal set of factors that do not include Myc or the transient delivery of the reprogramming factors by non-integrating methods. Now, four independent works report on genomic analyses of iPS and reveal a worrisome presence of mutations in these cells.”

One of the works mentioned is the March 2011 publication is Somatic coding mutations in human induced pluripotent stem cells.  “Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.”

A second relevant work is the May 2011 publication Genomic instability in induced stem cells. The work  “looks at the problem from a cancer-angle.  Given that oncogenes are known to generate a type of DNA damage known as replicative stress (RS) (Halazonetis et al, 2008), and that some of the reprogramming factors like c-Myc or Klf4 are known proto-oncogenes, they explored whether the reprogramming protocol could generate RS. In fact, a previous report had already shown that cells undergoing reprogramming presented a pan-nuclear phosphorylation pattern of histone H2AX, which is reminiscent of RS (Marion et al, 2009). In addition, DNA repair deficient cells show a poor reprogramming efficiency again, suggesting that some form of DNA damage could be generated during reprogramming. To evaluate this hypothesis, Pasi et al performed comparative genomic hybridization (cGH) analyses of iPS genomes. Their data show a significant number of chromosomal aberrations on iPS, which the authors suggest was in part influenced by the use of Myc. In fact, the authors report that whereas Myc is sufficient for the reprogramming of mammary progenitors into mammary stem cells, this protocol is accompanied by chromosomal abnormalities. Interestingly, this work revealed that the chromosomal rearrangements that occur during reprogramming frequently involved deletions mapping closely to known fragile sites, or to very large genes, supporting the concept that reprogramming could be accompanied by significant amounts of RS(ref).”

A third relevant 2011 paper is Copy number variation and selection during reprogramming to pluripotency.  “Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.”

A fourth relevant work is the January 2011 publication Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. This work argues that all pluripotent stem cells exhibit genomic instability.  As related by Blasco et al: “By performing a very comprehensive high-resolution SNP analysis of 189 pluripotent (iPS and ES) and 119 non-pluripotent samples, the authors found that the genomes of pluripotent cells are amazingly plastic, with frequent CNVs in pluripotency-related genes and pseudogenes. Noteworthy, the pattern of genomic aberrations was different in iPS or ES, again suggesting some intrinsic changes linked to the reprogramming process. The process of reprogramming led to small deletions, which included tumour suppressors, and which could be consistent with the idea of reprogramming-induced RS. However, time in culture led to the accumulation and selection of novel genomic aberrations in both iPS and ES, which were quantitatively of the same magnitude as those inflicted during reprogramming. This work illustrates the remarkable plasticity of pluripotent genomes and strongly suggests that the use of early passage lines should be an important factor to consider when working with pluripotent cells.”  Further. “For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety(ref).”

IPSCs tend to be oncogenic

The problem of mutations in iPSCs poses as serious challenge to the use of such stem cells for human therapeutic purposes.  The propensity of iPSCs to generate tumors has been known for some time.  For example, see the 2009 review article The tumorigenicity of diploid and aneuploid human pluripotent stem cells and the 2011 article Stem cells: The dark side of induced pluripotency.

Another 2011 review article The tumorigenicity of human embryonic and induced pluripotent stem cells reports “Until recently, it was assumed that human induced pluripotent stem cells (HiPSCs) would behave like their embryonic counterparts in respect to their tumorigenicity. However, a rapidly accumulating body of evidence suggests that there are important genetic and epigenetic differences between these two cell types, which seem to influence their tumorigenicity(ref).”
An April 2011 publication Intramyocardial transplantation of undifferentiated rat induced pluripotent stem cells causes tumorigenesis in the heart warns “Our study demonstrates that allogeneic iPSC transplantation in the heart will likely result in in situ tumorigenesis, and that cells leaked from the beating heart are a potential source of tumor spread, underscoring the importance of evaluating the safety of future iPSC therapy for cardiac disease.”

A June 2011 publication Dissecting the Oncogenic Potential of Human Embryonic and Induced Pluripotent Stem Cell Derivatives reports “In this study, we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells, and compared their transcriptomes with those of five different types of cancer. — Overall, our findings suggest that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation, and additional stringent functional assays to purify these cells should be performed before they can be used for regenerative therapy.”

Much is being learned about iPSCs.  There is a great deal of variability among iPSCs depending on cell type of origin and reprogramming method and typically, variable pluripotency unequal to that of hESC counterparts.

The May 2011 publication The transcriptional and signalling networks of pluripotency reports “Pluripotency and self-renewal are the hallmarks of embryonic stem cells. This state is maintained by a network of transcription factors and is influenced by specific signalling pathways. Current evidence indicates that multiple pluripotent states can exist in vitro. Here we review the recent advances in studying the transcriptional regulatory networks that define pluripotency, and elaborate on how manipulation of signalling pathways can modulate pluripotent states to varying degrees.”  This article gets into some of the exquisite detail involved in reversion to and maintenance of pluripotency.  “These studies additionally revealed that in mESCs many of the key pluripotency-associated factors (Oct4, Sox2, Nanog, Esrrb, Sall4, Dax1, Klf2, Klf4, Klf5, Stat3 and Tcf3) may autoregulate their own expression21, 22, 23, 24, 25, 26, 32, 33, 34. It is possible that certain transcription factors directly downregulate the transcription of their own genes to prevent overactivation of gene expression.  Overexpression of pluripotency-associated transcription factors has been shown to perturb the homeostasis of mESCs; for example, overexpression of Oct4 and Sox2 triggers differentiation16, 35. Hence, the continual activation of these genes may destabilize the mESC state. — Biological networks consist of highly connected nodes called hubs, which if removed would lead to fragmentation of the network. Some of the genes that constitute hubs receive extensive inputs. For example, the enhancer region of the Oct4 gene is bound by at least 14 transcription factors (Oct4, Sox2, Nanog, Sall4, Tcf3, Smad1, Stat3, Esrrb, Klf4, Klf2, Klf5, E2f1, n-Myc and Zfx), and the enhancer region of the Nanog gene is bound by at least 9 transcription factors (Nanog, Klf4, Klf2, Klf5, Sall4, E2f1, Esrrb, Stat3 and Tcfcp2l1; refs 23, 25, 36). These genomic sites serve as key contact points and represent the most crucial integration nexus within the transcriptional regulatory network. —  There is also a correlation between the level of occupancy of gene promoters and transcriptional status. Genes bound by more transcription factors tend to be more actively transcribed, whereas genes with low level of transcription-factor occupancy are silenced in mESCs23, 24.”Increased numbers of transcription-factor-binding datasets coupled with precise measurement of gene expression could provide a more sophisticated and integrated analysis to reveal the underlying rules of ESC-specific gene regulation and the combinatorial nature of transcription factor regulation37, 38, 39.”  Also, the article discusses the interface of the pluripotent transcription factor networks with histone modification, microRNAs and non-coding RNAs.  A chart only for action of the Oct4 gene is:

The article concludes: “Several stem cell lines with key characteristics of pluripotency have been derived from mammalian embryos. Although these stem cells express transcription factors (Oct4, Sox2 and Nanog) that are classically associated with pluripotency, there are substantial differences in the features of the transcriptional regulatory networks that characterize them. Deciphering these networks is likely to provide new mechanistic insights into the regulation of pluripotent states. It is also evident that transcription factors are powerful modulators of pluripotent states as they can induce the transition between different states. Many of the methodologies at hand to convert or induce pluripotent states involve the use of chemical inhibitors targeting specific signalling pathways, highlighting the importance of understanding the roles of signalling through extrinsic factors. Overall, the combinatorial use of transcription factors and chemical modulators will enable the development of new approaches to shape cellular states, and possibly create novel ones.”

The March 2011 publication Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells reports “Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.”

The November 2011 publication A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity reports identifying factors relating to pluripotency in hESCs that can be looked for also in iPSCs. “The derivation of human ES cells (hESCs) from human blastocysts represents one of the milestones in stem cell biology. The full potential of hESCs in research and clinical applications requires a detailed understanding of the genetic network that governs the unique properties of hESCs. Here, we report a genome-wide RNA interference screen to identify genes which regulate self-renewal and pluripotency properties in hESCs. Interestingly, functionally distinct complexes involved in transcriptional regulation and chromatin remodelling are among the factors identified in the screen. To understand the roles of these potential regulators of hESCs, we studied transcription factor PRDM14 to gain new insights into its functional roles in the regulation of pluripotency. We showed that PRDM14 regulates directly the expression of key pluripotency gene POU5F1 through its proximal enhancer. Genome-wide location profiling experiments revealed that PRDM14 colocalized extensively with other key transcription factors such as OCT4, NANOG and SOX2, indicating that PRDM14 is integrated into the core transcriptional regulatory network. More importantly, in a gain-of-function assay, we showed that PRDM14 is able to enhance the efficiency of reprogramming of human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Altogether, our study uncovers a wealth of novel hESC regulators wherein PRDM14 exemplifies a key transcription factor required for the maintenance of hESC identity and the reacquisition of pluripotency in human somatic cells.”

iPSCs can generate immune reactions even in individuals from which the source cells were taken

It has always been thought that iPSCs that are autologous to an individual (i.e. resulting from reprogramming of cells from an individual and then re-introduced into that same individual) would not initiate an immune system rejection response.  Surprisingly, not so!  At least, not so in our mouse cousins. The June 2011 publication Immunogenicity of induced pluripotent stem cells reports “Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells1, 2, 3, 4, 5. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.”

Incomplete epigenetic reversal appears to be a characteristic of iPSCs

A major reason why iPSCs generated through most known methods fail to exhibit full hESC-type pluripotency is that epigenetic markers of the source cell types are not completely wiped out.  The March 2011 publication Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells relates to this point.  “Human induced pluripotent stem (iPS) cells are remarkably similar to embryonic stem (ES) cells, but recent reports indicate that there may be important differences between them. We carried out a systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low-passage iPS cells analysed retain a transcriptional memory of the original cells. The persistent expression of somatic genes can be partially explained by incomplete promoter DNA methylation. This epigenetic mechanism underlies a robust form of memory that can be found in iPS cells generated by multiple laboratories using different methods, including RNA transfection. Incompletely silenced genes tend to be isolated from other genes that are repressed during reprogramming, indicating that recruitment of the silencing machinery may be inefficient at isolated genes. Knockdown of the incompletely reprogrammed gene C9orf64 (chromosome 9 open reading frame 64) reduces the efficiency of human iPS cell generation, indicating that somatic memory genes may be functionally relevant during reprogramming.”

Micro RNAs can be used for reprogramming to generate iPSCs

“Reprogramming of human somatic cells into induced pluripotent stem cells (iPSCs) was first achieved by ectopic expression of OCT4, SOX2, KLF4 and c-MYC or OCT4, SOX2, LIN28 and NANOG87, 88, 89. Using the same approach, Ding and colleagues infected human fibroblasts with OCT4, SOX2, NANOG and LIN28 to generate iPSCs90. Instead of using conventional hESC culture conditions, mESC medium with human LIF was then used to select the reprogrammed cells.  These cells were maintained with a combination of chemical inhibitors (MEK, ALK5 and GSK3 inhibitors)(ref).”  Subsequently a number of alternative approaches have been developed for reprogramming into iPSCs.  I reported on some of those approaches a little less than a year ago in the blog entry Induced pluripotent stem cells – developments on the road to big-time utilization.  One of the latest approaches involves the use of micro RNAs.

The April 2011 publication Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells reports “The embryonic stem cell–specific cell cycle–regulating (ESCC) family of microRNAs (miRNAs) enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells1. Here we show that the human ESCC miRNA orthologs hsa-miR-302b and hsa-miR-372 promote human somatic cell reprogramming. Furthermore, these miRNAs repress multiple target genes, with downregulation of individual targets only partially recapitulating the total miRNA effects. These targets regulate various cellular processes, including cell cycle, epithelial-mesenchymal transition (EMT), epigenetic regulation and vesicular transport. ESCC miRNAs have a known role in regulating the unique embryonic stem cell cycle2, 3. We show that they also increase the kinetics of mesenchymal-epithelial transition during reprogramming and block TGFβ-induced EMT of human epithelial cells. These results demonstrate that the ESCC miRNAs promote dedifferentiation by acting on multiple downstream pathways. We propose that individual miRNAs generally act through numerous pathways that synergize to regulate and enforce cell fate decisions.”

LincRNAs are are direct targets of key pluripotency transcription factors and involved in reprogramming to iPSCs

The December 2010 publication Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells reports: “The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome, resulting in altered patterns of gene expression. Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs) that occurs upon derivation of human iPSCs and identify numerous lincRNAs whose expression is linked to pluripotency. Among these, we defined ten lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells, suggesting that their activation may promote the emergence of iPSCs. Supporting this, our results indicate that these lincRNAs are direct targets of key pluripotency transcription factors. Using loss-of-function and gain-of-function approaches, we found that one such lincRNA (lincRNA-RoR) modulates reprogramming, thus providing a first demonstration for critical functions of lincRNAs in the derivation of pluripotent stem cells.”

A few observations

This has been a selective reporting on iPSC research published in the last seven months.  Even though I have covered a fair number of publications here, there is much additional ground that I might cover in later blog entries.  In particular I would like to provide an update on advances in other forms of cell reprogramming and, as I stated above, I plan soon to generate a subsequent blog entry devoted to the topic of longevity of stem cells and the roles stem cells play in human longevity. I will also do a blog post at some point on progress in directing iPSC and hESC differentiation into other somatic cell types.

The nature of stem cells of all kinds, the broad area of cell reprogramming, and the generation of iPSCs are topics on the cutting edge of research in biology.  The rate of research in these areas is accelerating mightily and a lot is being learned.

The complexity in these areas is extraordinary.  Involved are a large number of renewal genes, signaling pathways, transcription factors and co-factors, lincRNAs and microRNAs, pluripotency factors, histone acetylation and DNA methylation, other chromatin remodeling, pathways involving mitochondria, cell senescence and repair mechanisms and factors still being discovered..  I don’t think this should be too surprising.  We should not be impatient since what is being discovered, finally, is the nature of life itself.

We are probably close to the start of the journey which may take decades before we are reasonably near the end of understanding most what we really need to know to promote health and extend life.

For the present iPSCs are useful for in-vitro and animal studies and for modeling the effects of drugs(ref), but they are still beset with multiple problems identified above that means they cannot yet be used for human therapeutic purposes.  My vision of closing the loop in the stem cell supply chain remains as a possibility for the future but many obstacles must be overcome before that possibility matures into reality.

The last three weeks has seen a series of blog problems and a hiatus of new postings.  Now the last of these problems are being straightened out and I and my colleague Victor are getting back to serious writing. Here is what happened and where we are now:
1.         Having accumulated some 360 posts and a thousand comments, the blog ran out of allocated space in my web hosting service and no longer accepted any new postings.
2.       After significant prodding the web hosting service installed an updated version of the Blog software and ported our historical blog content to it.  This worked to increase our space but had two serious unintended consequences.
a.       First, the move attracted the attention of web comment spammers who started bombarding the blog with spam comments, as many as 260 a day.  This required launching a war on spam which I believe is now won,   See the blog entry Spam update.  If there is a remaining problem, I will take additional steps.  If you are a reader and have a problem with a legitimate comment, please e-mail me personally at vegiuliano@comcast.net.

b.  Second, past blog entries ported to the new software started to show up with Strange characters in blog posts.  The character mapping used in the blog conversion was faulty.  Obvious fixes applied by my hosting company did not work.  A loyal reader of this blog, Abhijit Mhapsekar has been extremely helpful in providing me with fixes that have so far taken care of 95% of the problem by correcting for the most frequently-found misinterpreted characters.  Abhijit and I have spotted a number of other instances of misinterpreted characters and are in the process of correcting for those as well.   Also, a few past entries show up now with double line spacing.  However, all past blog posts remain essentially readable.

3.       These technical glitches have consumed much of my time so there have been no substantive new blog postings since May 23.  This situation should change soon.  I am working on a new posting which will be an Update on cell reprogramming and induced pluripotent stem cells which should be available in a day or two and I understand that Victor is also working on a new post.
Thank you for y our patience during this transition period.  Despite these issues at least a dozen new legitimate blog registrations are coming every day and blog traffic continues to increase with an average of about 2,000 unique users accessing an average of 1.7 blog entries every day.  Here is to the long haul, both for increasing human longevity and for increasing the life of this blog!

Vince

By Vince – admin

Our blog has been under a massive robot spam comment attack over the last four days.  We received, I estimate, 500 comments and all but a few were spam.  These inundated my personal mail and attached themselves to a number of past blog entries. The response to yesterday’s  announced New Policy regarding spam comments was an intensification of the attack.  So, I have taken additional measures.

*  New comments must now be approved before they are posted.

*  A spam-detection and filtering engine is now in place.

*   I have deleted hundred of comments in the blog that appeared to me to be spam, though many more probably still remain yet-undetected.  I am continuing to review past comments and delete ones that seem to be spam.

Deleting spam entries requires judgment calls and my judgments may not have all been correct.  When I see complementary comments about the blog, I often can’t tell if is a real person or a spam robot talking.   I have deleted very many but not all general complements that could apply to any blog, comments like “Thank you for your good work.  You have done a careful job in writing this blog and the information you have provided has been valuable for me.” Sometimes I saw a seemingly thoughtful comment, but then found that that the same comment appeared in several places.  Only then did I know it was spam.

So I have to apologize first for any sincere user comments that I eliminated as spam, and second for all spam that I gave a pass to.  If I spammed your sincere comment, please submit it again with a little more detail about the related post so I know it is real.  If you recognize something you know is spam, let me know so I can zap it.

Vince

From Vince, Admin

After we switched to the new blog software, many past blog entries show up with strange interspersed characters such as at the end of sentences and †where an apostrophe (‘) should be.  Although these symbols are annoying the blog entries are still by-and-large readable.  Rather than going back to correct 360 or so past entries, I am asking my web hosting company to see if they can fix the problem.  My guess is that it is simply a matter of a corrupted symbol translation table.  So, please have patience as we go through the rest of this transition.

Vince

From Vince, Admin

This blog has been receiving a great many spam comments recently.  I deleted a dozen last night alone and there are a dozen new ones now.  I suspect that we are being targeted by a spam company. So, I have to announce strong measures to protect the integrity of our content.  Some of the spam mail is obvious product advertising , often having nothing to do with health or longevity.  There is also a more subtle kind of spam, from “users” which are actually hyperlinks to commercial selling websites.  Recent examples are bed liner, victoria secret bathing suit, russian women for marriage, gold exchange rate today and hormone replacement therapy . The content of the spam in such cases may be neutral or and is often complementary such as saying “Nice blog.  Keep up the good work.”  I cannot tell if some such comments are sincere but I strongly suspect most of them are spam, designed to enhance search engine placements for the selling sites concerned.  Thousands of users visit this blog daily and it is indexed daily by major search engines like Google.  The more times a link appears on an indexed site like this one, the theory goes, the higher the search index placement for the link.  So spam companies are selling services to websites “to improve your search engine placement” which means massive spamming of blogs like ours.

So, here is the new policy:

1.   Comments that are clearly irrelevant to the content of the related post will be deleted.

2.  Comments that are aimed at promoting commercial products or services will be deleted, even if the products might relate to health or longevity.

3.   Comments from users that are commercial sites will be treated with suspicion and unless particularly relevant to the post concerned will be deleted.  I request users to sign up with names that are not commercial sites.

This policy will go into effect immediately and I will be reviewing past comments and applying the policy to them as well.  I continue to welcome serious comments that contribute to the content of this blog. Please, if you want to praise or acknowledge the blog, sign up as an individual user to do so, not as a selling site.  And if you see something advertised in a comment I have not managed to delete, don’t buy it. I hope not to have to take more drastic measures restricting comments but, if necessary, will.

If  you are operating a spam company, please LEAVE US ALONE.  Our purpose here is communication of scientific information.  I could probably make a fair amount of money selling advertising but have decided not to.  So, NO HITCH-HIKE ADVERTISING.

Vince`

We have been having trouble with the blog software for the last few months – our blog was too big for it with 365 posts and 969 comments.  Our hosting company has provided us with a new dedicated WordPress software installation which should keep us going for some time more.

Do you like the new presentation format?  It makes better use of screen real estate with a wider reading format.  All the old material is still here, rest assured.

Vince

By Victor

One of the paradoxes of longevity research has been the observation that persons of exceptional longevity actually have more disease-related genes in their genome than ordinary people.  Intuitively, one would expect just the opposite, that the lack of disease-related genes would explain their increased lifespans.  Although they have a higher than normal incidence of genes known to be associated with disease conditions, they appear to have some kind of innate protection against the effects of those genes.  A hypothesis explaining this apparent paradox was proposed, and experimentally validated in a study of Ashkenazi Jewish centenarians who participated in the Longevity Gene Study at the Albert Einstein College of Medicine.  Apparently, certain longevity genotypes “buffer” or cancel out the effects of harmful genes, allowing them to accumulate.

Buffering Mechanisms in Aging: A Systems Approach Toward
Uncovering the Genetic Component of Aging:

“We corroborated this hypothesis by studying gene–gene interactions between age-related disease genotypes and longevity genotypes.    Our findings suggest that individuals with the favorable longevity genotype can have just as many deleterious aging genotypes as the rest of the population because their longevity genotype protects them from the harmful effects of the other. We identify genes contributing to extreme lifespan as well as their counterpart, age-related disease genesAs an example of this buffering mechanism, they found, for instance, that a SNP of the CETP gene I405V protected against the effects of disease-causing LpA gene.  SNP, or Single Nucleotide Polymorphism means that an amino acid substitution is found in a single base-pair of a gene.  In this case, isoleucine is substituted for valine in codon 405 of the gene encoding CETP (Cholesterol Ester Transfer Protein).  This genetic variant is referred to as CETPVV when individuals are homozygous, which means that both chromosomes have the same SNP.  LpA is the gene for a lipoprotein that predisposes carriers for heart disease.(ref)  It was previously known that CETPVV was associated with exceptional longevity.(ref)  Individuals with CETPVV have abnormally large lipoprotein particles (both HDL and LDL); and they have lower rates of cardiovascular disease and metabolic disorders.  They also have lower rates of cognitive decline.(ref)  From a more recent study published in the JAMA in 2010:

“We found that people with two copies of the longevity variant of CETP had slower memory decline and a lower risk for developing dementia and Alzheimer’s disease,” says Amy E. Sanders, M.D., assistant professor in the Saul R. Korey Department of Neurology at Einstein and lead author of the paper. “More specifically, those participants who carried two copies of the favorable CETP variant had a 70 percent reduction in their risk for developing Alzheimer’s disease compared with participants who carried no copies of this gene variant.” (ref)

Will the next generation of CETP inhibitors provide protection from cognitive decline and increased lifespan, as well as protection from cardiovascular disease?

Not necessarily.  Remember the longevity gene CETPVV does not prevent expression of CETP; it alters the expression of CETP in ways that are not yet fully understood, and may be difficult to mimic pharmaceutically.  Some type of gene therapy which inserts the CETPVV variant, using an adenovirus or stem cells might be a possible alternative treatment strategy.  This transport protein is known to play a vital role in both the intra- and extra-cellular transport of lipids.  For example, cholesterol and triglycerides are synthesized within the endoplasmic reticulum; however, they are metabolized and stored in other cellular compartments.  Evidence suggests that by interfering with the normal intracellular transport of lipids, inhibiting CETP could cause abnormal intracellular fat distribution accompanied by ysfunctional lipid metabolism and energy storage.  Fat cells are not merely fat storage depots, but actively secrete many signaling hormones, known as “adipocytokines.”  Abnormal adipocytokine secretion is associated with numerous health problems, including heart disease, diabetes, obesity, etc.

Possible Role for Intracellular Cholesteryl Ester Transfer Protein in Adipocyte Lipid Metabolism and Storage:“The newly discovered secretory functions of adipocytes have shifted the view of adipose tissue from being a simple energy storage tissue to one where this tissue functions as a major endocrine organ. In addition to their cholesterol and TG storage function, adipocytes also synthesize and secrete a variety of factors, such as leptin, adiponectin, angiotensinogen, resistin, and lipoprotein lipase, that regulate whole body energy balance and lipid homeostasis (59, 60). The secretion of these factors is closely linked to the lipid status of adipocytes. Both hypertrophy (excess of lipid content) and hypotrophy (low lipid content) of adipocytes have been shown to disrupt the secretion of these factors and cause abnormal whole body metabolism and inadequate insulin responsiveness (59, 60). Our studies demonstrate that CETP deficiency leads to abnormal TG and cholesterol storage and lowers the membrane ratio of free cholesterol/protein, factors reported to be associated with induction of insulin resistance and alteration in the synthesis of adipocytokines (44). Our findings, if they can be extrapolated to adipose tissue, suggest an important role for CETP in regulating the multiple functions of adipocytes.”

For a more in-depth discussion of CETP longevity variants, see Dr. Guiliano’s earlier entry:  CETP Gene Longevity Variants.

• Quantum coherence and its interplay with protein environments in photosynthetic electronic energy transfer (2010),

• Evidence for wavelike energy transfer through quantum coherence in photosynthetic systems (2007),

• Dynamics of light harvesting in photosynthesis. (2009)]

Drug discovery